Refinement of noncalorimetric determination of the change in heat capacity, Δ C p , of protein unfolding and validation across a wide temperature range

The change in heat capacity, ΔC p , on protein unfolding has been usually determined by calorimetry. A noncalorimetric method which employs the Gibbs‐Helmholtz relationship to determine ΔC p has seen some use. Generally, in this method the free energy change on unfolding of the protein is determined at a variety of temperatures and the temperature at which ΔG is zero, T m , and change in enthalpy at T m are determined by thermal denaturation and ΔC p is then calculated using the Gibbs‐Helmholtz equation. We show here that an abbreviated method with stability determinations at just two temperatures gives values of ΔC p consistent with values from free energy change on unfolding determination at a much wider range of temperatures. Further, even the free energy change on unfolding from a single solvent denaturation at the proper temperature, when coupled with the melting temperature, T m , and the van't Hoff enthalpy, ΔH vH , from a thermal denaturation, gives a reasonable estimate of ΔC p , albeit with greater uncertainty than solvent denaturations at two temperatures. We also find that nonlinear regression of the Gibbs‐Helmholtz equation as a function of stability and temperature while simultaneously fitting ΔC p , T m , and ΔH vH gives values for the last two parameters that are in excellent agreement with experimental values. Proteins 2008. © 2008 Wiley‐Liss, Inc.